Primordial growth failure has been linked to defects in the biology of cell division and replication. The complex processes involved in microtubule spindle formation, organization and function have emerged as a dominant patho-mechanism in these conditions. The majority of reported disease genes encode for centrosome and centriole proteins, leaving kinetochore proteins by which the spindle apparatus interacts with the chromosomes largely unaccounted for. We report a novel disease gene encoding the constitutive inner kinetochore member CENPT, which is involved in kinetochore targeting and assembly, resulting in severe growth failure in two siblings of a consanguineous family. We herein present studies on the molecular and cellular mechanisms that explain how genetic mutations in this gene lead to primordial growth failure. In both, affected human cell lines and a zebrafish knock-down model of Cenpt, we observed aberrations in cell division with abnormal accumulation of micronuclei and of nuclei with increased DNA content arising from incomplete and/or irregular chromosomal segregation. Our studies underscore the critical importance of kinetochore function for overall body growth and provide new insight into the cellular mechanisms implicated in the spectrum of these severe growth disorders.
Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10 4 muscle SP cells or with 10 6 MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells.
Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G ‐protein coupled receptor 56 ( GPR 56) was transiently up‐regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR 56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR 56 in skeletal muscle. GPR 56 −/− myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR 56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T ‐cell ( NFAT )c2. Our data suggest that these abnormalities result from decreased GPR 56‐mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR 56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild‐type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR 56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR 56 knockout mice and human patients suggests that other factors may compensate for the lack of this G ‐protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities.
Applications of PCR have revolutionized the field of immunogenetics particularly in studies of human leukocyte antigen class II polymorphism and more recently in the analysis of T cell receptor usage. The diversity of the variable region of the T cell receptor, however, has made it difficult to amplify the complete repertoire of T cell receptor transcripts. We have chosen to address this problem through the design of oligonucleotide primers specific for each of the known V alpha- and V beta-region T cell receptor families in order to characterize the T cell receptor repertoire. Using nonradioactive probes labeled with horse radish peroxidase, the system presented here allows for the rapid elucidation of the T cell receptor repertoire expressed in cells or tissue samples, such as those derived from autoimmune lesions. The identification of the T cell receptor repertoire involved in a pathogenic process can have therapeutic implications given the success of reversing experimental autoimmune disorders by directing specific forms of immunotherapy against V region gene products.
Skeletal muscle cells can be used in vitro for the study of myogenesis, as well as in vivo as gene-delivery vehicles for the therapy of muscle and nonmuscle diseases. These skeletal muscle cells are derived from muscle satellite cells that lie between the basal lamina and the sarcolemma of differentiated muscle fibers (1). Normally quiescent after the period of muscle development and growth during fetal life and the early postnatal period, these cells are induced to proliferate upon muscle damage and fuse with existing muscle fibers. Satellite cells isolated and grown in vitro are called myoblasts. Myoblasts proliferate in mitogen-rich media, but upon reaching high cell density followed by exposure to mitogen-poor media, are induced to differentiate and become postmitotic. Muscle differentiation is characterized by the fusion of myoblasts to form multinucleated myotubes that express differentiationspecific proteins. In this chapter, methods are given for the isolation of myoblasts from human muscle tissue using two different techniques: (a) flow cytometry (2) and (b) cell cloning (3,4). Recent reports have also highlighted the existence of highly primitive cells within mouse skeletal muscle, whose relationship with satellite cells is still under study (5-7). These primitive cells have been purified using different methods and techniques, including the preplating technique (8-10) and the fluorescence-activated cell sorter (FACS) (11-14). Depending on the isolation technique, these cells have been named differently. Muscle SP cells have been isolated from mouse skeletal muscle by staining the dissociated primary muscle cells with the vital DNA dye Hoechst 33342, followed by FACS purification (11-14). Mouse muscle SP cells have demonstrated hematopoietic and myogenic differentiation potential both in vitro and in vivo (11-14) and these studies are being extended to human-derived SP cells. Methods to isolate human muscle SP cells from fetal and from adult skeletal muscle are also given. The methods in this chapter are applicable to muscle tissue from both fetal and postnatal donor as well as from normal and diseased individuals.
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